Abnormalities in human limb skeletal development are one of the most common human birth defects, yet little is known as to the cellular changes underlying these congenital malformations. Many of the studies on limb skeletal development in vertebrate model organisms have explored the function of a relatively small set of patterning genes. However, there are many gaps in our knowledge as to how the activity of these genes control the dynamic cellular events that lead to the formation of cartilage elements of the correct number, shape, and size. A major limitation to a deeper understanding of this process resides in the fact that cartilage morphogenesis has historically been examined in fixed and stained specimens at static and infrequent intervals. To help close these gaps in our knowledge, we have developed a unique live cell imaging approach for cartilage formation to dynamically visualize limb mesenchymal progenitor cells as they form mesenchymal condensations that subsequently differentiate into cartilage. This approach has identified novel cellular events that are critically required for the formation of a cartilage template, controlled by key molecular regulators found mutated in human limb and chondrodysplasia syndromes. We have also made advances in the development of novel strategies to image living intact vertebrate embryos (mouse and chick) at a single cells resolution, allowing us to visualize the full interplay of cell and tissue dynamics during cartilage development that has not been previously possible to resolve. Utilizing this imaging technology, we have identified a previously unrecognized mode of cell-to-cell communication that occurs via novel cytoneme-like cytoplasmic extensions that we have termed vertebrate cytonemes (v-cytonemes) present on limb mesenchyme, which appear to direct cartilage formation. Such cytoplasmic extensions, extending many cell diameters in length, can only be visualized in living but not fixed tissue and have thereby never been previously observed on mesenchymal progenitor cells in vivo. Our preliminary findings suggest that a network of cytoplasmic extensions connect signaling centers and traffic signaling components in the vertebrate limb bud to direct the formation of a cartilage template. In Aim1 we wil determine the landscape and orientation of v- cytonemes within the developing limb bud in-vivo. These studies will also be complemented by direct tests of v-cytoneme function by key ligands, Shh and Fgf(s), which pattern the limb cartilage template. In Aim2 we wil test the hypothesis that Shh movement through v-cytonemes present on mesenchymal cells acts as a mechanism for long-range cell signaling. In Aim3 we will systematically delineate the components of the Shh signaling pathway that localize and employ v-cytonemes as a mechanism for long range signaling. The imaging technology and studies described in this proposal wil open a portal into a previously unexplored area: the dynamics of cell-to-cell signaling, visualized at a single cell level, leading to the formation of a cartilage template.